Assessing hASC Viability and Growth

hASCs, seeded in a 48-well plate (Corning Incorporated, Corning, NY, USA), were exposed for 24, 72 h (3 days), 6 or 14 days to CB or DMSO. Moreover, hASC recovery was assessed after 24 or 48 h from the end of 24 h CB treatment. At each time point, cells were detached (2 wells/condition for each experimental point) by trypsin–EDTA and counted, as previously described [45 (link)]. Briefly, cells were resuspended in a medium with 50% erythrosine B (Sigma-Aldrich Co., St. Louis, MO, USA) and 0.2% red dye in PBS. Not stained viable cells and red stained dead cells were manually counted, at least twice for each condition, using the Neubauer hemocytometer (BRAND GmbH, Wertheim, Germany) and a light microscope (Nikon Eclipse TS100, Nikon Instruments, Melville, NY, USA). The total numbers of viable and dead cells were calculated according to the manufacturer’s instructions; for each sample, cell viability was obtained calculating the percentage of living cells compared to the total number of cells. Moreover, cell growth rate (gr) was calculated using the following formula: gr = (ln (N(t)/N(0))/t, where N(t) = the number of cells counted after 24 h or 48 h recovery upon CB removal; N(0) = the number of cells counted after 24 h of CB treatment; t = time passed (24 or 48 h).